Pertussis, caused by Bordetella pertussis remains a considerable economic and health burden in the USA, despite very high coverage of vaccination. Current pertussis vaccines although effective at preventing the severe form of disease do not prevent the carrier state. Adult and adolescent carriers are responsible for the transmission of the bacterium to infants and young children, in whom the disease is severe and sometimes lethal. There is an urgent need to identify new factors that will be protective against bacterial carriage and will be included in the acellular vaccine. It is hypothesized that nasopharyngeal carriage of B. pertussis in humans is due to its ability to exist in a sessile form known as biofilms. Our preliminary results demonstrate that B. pertussis exists in mice as multicellular community adherent to the nasal epithelium, which are reminiscent of biofilms. The overall objectives of this research are to study the role of the biofilm-associated Bps polysaccharide in biofilm development and pathogenesis and to gain insight into the regulatory circuitry that controls bps expression in biofilms and in the respiratory tract. Bps is similar to PGA/PIA/PNAG (poly-2-1,6-N- acetylglucosamine) polysaccharides of other bacteria including a number of pathogens. In Specific Aim 1, biologically relevant in vitro model systems will be utilized to study the mechanistic basis of the role of Bps in biofilm formation. The importance of Bps in providing protection from anti-microbial compounds will be examined by testing its role in conferring resistance against antibiotics, antimicrobial peptides and reactive oxygen species. It is hypothesized that Bps mediates survival and persistence of B. pertussis in the respiratory tract by playing an immunomodulatory role and by targeting one or more components of the immune system. Mouse models of B. pertussis intranasal infection will be employed to test these hypotheses. In Specific Aim 2, the establishment of temporal and spatial expression gradient of bps during biofilm formation and in the respiratory tract will be examined by real time RT PCR assays and measurement of GFP fluorescence. Finally, to provide insights into the complex networks that operate to control bps expression, the role of a regulatory protein BpsR in controlling the expression of the bps locus in biofilms and in the mouse respiratory tract will be examined. Relevance to public health. This project will provide a better understanding of the pathogenic mechanisms and the infectious cycle of B. pertussis in mammalian hosts. Our studies will stimulate the development of Bps-based vaccines and reagents like antibodies for treatment of pertussis. Since Bps-like polysaccharides are produced by a number of pathogens like Staphylococci, E. coli and Yersinia Spp, and possibly others, our results will contribute towards the treatment and the understanding of a wide variety of bacterial infections.